Project Summary. Zika virus (ZIKV; Flavivirus) is transmitted to humans by the mosquito vector Aedes aegypti and poses significant health risks to the Americas (1, 2).This phase I project from Colorado State University (CSU; Olson and Black), University of Missouri (MU; Franz) and University of Notre Dame (UND; Fraser) will apply gene drive technology to generate Ae. aegypti populations refractory to ZIKV and other arboviruses transmitted by this vector species. Our gene drive approach is based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 for rapid, effective, genetics-based population modification of Aedes aegypti. Our principal goals are: 1) apply two validated Anti-Viral Trans Gene (AVTG) strategies that target ZIKV to minimize or ablate ZIKV infection of Ae. aegypti, 2) generate transgenic Ae. aegypti that express the AVTGs in the context of a CRISPR/Cas9 (CC) gene drive system, 3) show that the genetically modified mosquitoes have a stable, refractory phenotype for ZIKV infection, and 4) validate gene drive (in indoor cage studies) by introgressing the AVTGs into an Ae. aegypti population to convert the population from a competent to a refractory infection phenotype. Previously, Drs. Olson and Franz have genetically modified Ae. aegypti (with no drive) that stably express DENV2-specific, inverted repeat (IR) RNAs forming dsRNA to trigger the small interfering (si)RNA pathway of RNAi in the mosquito (3, 4). Dr. Fraser has developed mosquito cell lines that express group I intron (GrpI) ribozymes targeted to a highly conserved region of arboviral genomes. He has already developed GrpI ribozymes that target all four DENV serotypes and chikungunya virus (CHIKV; Alphavirus) (6, 7). In this proposal, we will initially develop siRNA- and ribozyme-based anti-viral genes to target ZIKV in the context of CC gene drive. We will compare the two AVTG strategies with CC gene drive to see which is the most effective at suppressing mosquito infection. If the GrpI AVTG genes successfully suppress vector competence and spread the ZIKV AVTG into caged populations of Ae. aegypti, we will use other GrpI AVTGs with the CC drive system to additionally target DENVs and CHIKV. CC gene drive has already been used to efficiently modify cage-populations of Anopheles stephensi with anti- pathogen genes (8). We are confident we can adapt this approach to drive antiviral IR RNA and ribozyme AVTG genes to greatly reduce or ablate Ae. aegypti competence for transmitting ZIKV. The CC-AVTG system we propose will target two Ae. aegypti genome sites (TIMP P4 on chromosome aem 2q and 3'UTR polyadenylate binding protein (PABP on arm 3q). We use the ?2-tubulin promoter to express Cas9 to achieve gene drive through the male germ-line. This will maximize homology-dependent repair (HDR) to maintain drive over non-homologous end-joining (NHEJ) which impairs drive (9). This proposal represents a significant step towards developing and optimizing CC drive with AVTGs in Ae. aegypti. This effort will be foundational for using CC drive in Ae. aegypti for disease control We are confident the project greatly advances population replacement as a tool for potentially mitigating arbovirus transmission.